The mechanism of an inhibitory antibody on TF-initiated blood coagulation revealed by the crystal structures of human tissue factor, Fab 5G9 and TF.G9 complex.
The tissue factor (TF)-initiated blood coagulation protease cascade can be greatly inhibited in vivo by a potent anti-human-TF monoclonal antibody, 5G9. This antibody binds the carboxyl module of the extracellular domain of TF with a nanomolar binding constant and inhibits the formation of the TF.VIIa.X ternary initiation complex. We have determined the crystal structures of the extra-cellular modules of human TF, Fab 5G9, and their complex (TF.5G9) to 2.4 A, 2. 5 A, and 3.0 A, respectively, and measured the apparent inhibition constants of 5G9 on a panel of TF mutants. In our unliganded TF structure, a 7 degrees change in the relative orientation between the D1 and D2 modules was observed when compared with other published TF structures. Comparison of the free and bound Fab 5G9 indicates that small segmental and side chain variation of the antibody complementarity determining regions occurred on complexation with TF. The antibody-antigen recognition involves 18 TF antigen residues and 19 Fab residues from six CDR with one of the largest buried surface areas seen to date. A combination of structural and mutagenesis data indicate that Tyr156, Lys169, Arg200, and Lys201 play the major role in the antibody recognition. The TF. 5G9 structure provides insights into the mechanism by which the antibody 5G9 inhibits formation of the TF.VIIa.X ternary complex.
Huang
M
M
Department of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92027, USA.
Syed
R
R
Stura
E A
EA
Stone
M J
MJ
Stefanko
R S
RS
Ruf
W
W
Edgington
T S
TS
Wilson
I A
IA
D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D001777:Blood Coagulation / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry*; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D013925:Thromboplastin / Q000737:chemistry*
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D013925:Thromboplastin